Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

This method can help answer key questions in the pulmonary diseases research such as pneumonia, asthma, cystic fibrosis, and chronic obstructive pulmonary disease. The main advantage of this technique is to isolate and enrich patient neutrophil from patient airway secretion without vertical chemical disturbance. Demonstrating the procedure will be Kyungyong, a PhD student from my laboratory.

To start with, maintain a ratio of 10 to one and mix the PDMS precursor with the base and curing agent. Next, add 30 grams of the PDMS mixture on the micro machine aluminum mold. Then add another 20 grams of PDMS mixture on a 100 millimeter Petri dish.

For the purpose of degas, put the mold and the Petri dish in the vacuum desiccator. After degassing, release the vacuum and place the mold and the Petri dish in an oven at 90 degrees Celsius for an hour. After one hour, remove the mold and the Petri dish from the oven and let it cool at room temperature for 10 minutes.

Next, use a knife to cut the outline and punch the fluid access holes with two millimeter biopsy punch on the device. After punching the holes, secure a tape and adhere it to the channel side of the device and on the thick film of PDMS. Use forceps to peel the tape carefully to remove the dust.

Then start treating the channel side of the device and the plain PDMS with air plasma. Then bond both the channel side and the plain PDMS with the prepared supporting layer of plain PDMS. Next, leave the chip on a glass slide with dimensions of 102 by 76 millimeters.

Place the glass slide in an oven at 70 degrees Celsius for at least 30 minutes to increase the bond strength. Use a 10 milliliter syringe to collect one milliliter of airway secretion samples in nine milliliters of phosphate buffered saline. Then with the help of a blunt pipette, homogenize the mucus sample.

Next, use a 40 micrometer nylon cell strainer to strain the diluent and remove large chunks of tissue or blood clots. After the straining is complete, put the sample on ice. Then add 50 milliliters of phosphate buffered saline to the 0.5 milliliter sample diluent to achieve a final concentration of 1, 000 times diluted sample.

Use the fluid guide to assemble the PDMS chip in order to apply uniform flow to each of the four spiral micro channels. Next, use a male Luer connector with 1/16 inch diameter to connect to the inlet, the inner wall outlet, and the outer wall outlet ports of the fluid guide. Then connect the silicone tubing to the sample suspension.

Next, connect the peristaltic pump to the inlet tubing. Place the outer wall outlet tubing in the waste reservoir. Then position the end of the inlet tubing and the inner wall outlet tubing in the sample reservoir.

After positioning the tubing, leave a 50 milliliter tube filled with five milliliters of phosphate buffered saline without calcium and magnesium in the sample reservoir. Then in order to prime the device, start pumping at a flow rate of approximately one milliliter per minute. After placing the sample in the sample reservoir, switch on the peristaltic pump and set the flow rate at four milliliters per minute.

After the sample volume reaches the desired volume of approximately one to two milliliters, stop the operation and disconnect the tubing. Clinical sample consisting of patient tracheal secretion is used for the microfluidic dissociation method in order to obtain the PMN rich population of cells. On subjecting the microfluidic dissociation method, highly purified CD45, CD66B plus rich PMN population of cells are obtained.

Here, the samples obtained from two different patients represented as case one and two are subjected to microfluidic dissociation to obtain the resulting cell suspension. Next, on subjecting the patient airway secretion sample to microfluidics, 94%of PMN are recovered consistently from all six samples. On the other hand, subjecting the patient sample to DTT mucolysis method resulted in recovering only 53.5%of PMNs with wide variation among the samples.

The neutrophils isolated by the microfluidics and DTT mucolytic methods are treated with PMA to trigger the neutrophil elastase release. Interestingly, patient samples subjected to microfluidics show intact functional cells with increased release of elastase by PMA stimulation. Whereas cells obtained after mucolytic dissociation show no significant increase in the elastase release from six different patient samples studied.

This is due to a chemically disturbed neutrophil surface. Once mastered, this technique can be done in 50 minutes if it is performed properly. By providing a dissociation protocol to capture neutrophils from clinical airway secretions, the method presented in this study is expected to expand the scope of clinical research on respiratory diseases such as pneumonia, asthma, cystic fibrosis, and chronic obstructive pulmonary disease.

The implications of this technique extend towards therapy of immune related pulmonary diseases such as asthma and chronic obstructive pulmonary disease because the proposed methods provides a way to capturing patient immune cell in noninvasive and label-free manner. Though this method can provide insight into respiratory research, it can also be applied to other patient biofluids with challenging biometrics such as nasal fluid, cervical fluid, and intestinal fluid. After watching this video, you should have a good understanding of how to separate neutrophils from patient airway secretion using close-looped spiral inertia microfluidics.

Don't forget that working with clinical samples can be extremely hazardous. All experiments must be performed under a biosafety cabinet with the personal protective equipments.